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  • C12N15/09—Recombinant DNA-technology

Definitions

• the present invention relates to a yeast highly producing glutathione, which is used for pharmaceutical products, foods and the like, and a method of producing glutathione by using the yeast.
• Non-patent document 1 Nature Chemical Biology 9, 119-125 (2013 )
• the present invention aims to provide a yeast that is genetically modified so as to more highly produce glutathione, and a method of producing glutathione utilizing the yeast.
• the present inventors made intensive studies and found that an intracellular amount of glutathione including oxidized glutathione was increased in a yeast strain in which thiol oxidase activity was increased.
• glutathione reductase which is involved in reduction of oxidized glutathione
• a remarkable increase in glutathione production namely a significant increase in intracellular glutathione amount (oxidized glutathione + reduced glutathione) was revealed, which resulted in the completion of the present invention.
• glutathione production can be more synergistically improved by increasing the activities of ⁇ -glutamylcysteine synthetase (GSH1) and/or glutathione synthetase (GSH2) and/or glutathione transport enzyme (YCF1) in the thiol oxidase activity-increased strain.
• GSH1 ⁇ -glutamylcysteine synthetase
• GSH2 glutathione synthetase
• YCF1 glutathione transport enzyme
• the present invention relates to a method of producing glutathione characterized in culturing a yeast whose thiol oxidase activity is increased.
• the present invention provides the following.
• glutathione can be efficiently produced.
• the present invention is a method of producing glutathione by a yeast in which intracellular thiol oxidase activity is increased, and preferably, glutathione reductase activity is reduced.
• enzyme activity is increased means that an enzyme activity of interest is increased as compared to that of the parent strain such as wild-type strain and the like.
• Enzyme activity is increased encompasses not only increasing an enzyme activity of interest in a strain natively having the enzyme activity, but also conferring an enzyme activity of interest to a strain natively lacking the enzyme activity.
• Increase of enzyme activity can be accomplished by, for example, artificially modifying a gene of a strain. Such modification can be achieved by, for example, enhancing the expression of a gene encoding an enzyme of interest.
• Enhancement of gene expression can be achieved by, for example, substituting the promoter of the gene on chromosome with a more potent promoter.
• the "more potent promoter” means a promoter that improves transcription of a gene as compared to the naturally occurring wild-type promoter.
• a more potent promoter a highly active form of native promoter may be obtained using various reporter genes.
• known high expression promoters for example, PGK1, PDC1, TDH3, TEF1, HXT7, ADH1 and the like gene may also be used.
• the substitution with a more potent promoter can be utilized in combination with the below-mentioned increase of copy number of gene.
• a method of enhancing ⁇ -glutamylcysteine synthetase activity by substituting the promoter of the ⁇ -glutamylcysteine synthetase gene on chromosome with a promoter having a potent transcriptional activity is disclosed ( Yasuyuki Ohtake et al., Bioscience and Industry, 50(10), 989-994, 1992 ).
• Enhancement of gene expression can be achieved by, for example, increasing copy number of the gene.
• the copy number of a gene can be increased by introducing the gene of interest onto the chromosome.
• Introduction of a gene onto the chromosome can be performed, for example, by utilizing homologous recombination.
• many copies of a gene can be introduced into the chromosome by homologous recombination using a sequence containing many copies in the chromosome as the target.
• sequence containing many copies in the chromosome autonomously replicating sequence (ARS) consisting of unique short repeat sequences, and rDNA sequence having about 150 copies can be mentioned.
• ARS autonomously replicating sequence
• yeast was transformed as described in WO 95/32289 .
• a gene may be incorporated into a transposon, and the transposon may be transferred into the chromosome to introduce many copies of the gene.
• the copy number of a gene can also be increased by introducing a vector containing the gene of interest into a host.
• a vector for example, a plasmid having a replication origin of CEN 4 or a multiple copy type plasmid having a replication origin of 2 ⁇ m DNA can be used preferably.
• the gene of interest may be inserted into a vector in combination with a suitable promoter to achieve expression of the gene of interest.
• the gene of interest may be expressed by utilizing the promoter in the vector.
• a modification to increase enzyme activity can also be achieved by, for example, enhancing the specific activity of the enzyme of interest.
• An enzyme having an enhanced specific activity can be obtained by, for example, searching through various organisms.
• a highly active form may be acquired by introducing mutation into native enzymes.
• the specific activity may be enhanced solely or enhanced in free combination with the above-mentioned method for enhancing gene expression.
• Increase in the enzyme activity of interest can be confirmed by measuring the activity of the enzyme.
• Thiol oxidase activity can be measured by the method described in FEBS,Letters 477 (2000) 62-66 .
• ⁇ -Glutamylcysteine synthetase activity can be measured by the method of Jackson ( Jackson, R. C., Biochem. J., 111, 309 (1969 )).
• Glutathione synthetase activity can be measured by the method of Gushima et al. ( Gushima, T. et al., J. Appl. Biochem., 5, 210 (1983 )).
• Glutathione transport enzyme activity can be measured by reference to THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 50, Issue of December 11, pp. 33449-33454, 1998 .
• Increase of the transcription amount of a gene encoding the enzyme of interest can be confirmed by comparing the amount of mRNA transcribed from the gene with that of the parent strain.
• a method for evaluating the amount of mRNA Northern hybridization, RT-PCR and the like can be mentioned ( Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001 )).
• a preferable increase in the amount of mRNA is, for example, not less than 1.5-fold, not less than 2-fold, or not less than 3-fold, as compared to the parent strain.
• the yeast of the present invention may heterozygously have a chromosome modified to increase enzymatic activity and a wild-type chromosome, or may be a homozygote of a chromosome modified to increase enzymatic activity, as long as it can accumulate glutathione.
• Decrease of enzyme activity can be accomplished by, for example, artificially modifying a gene of a strain. Such modification can be achieved by, for example, mutagenesis treatment or genetic recombination technique.
• a modification that decreases the enzyme activity can be achieved by, for example, decreasing the expression of a gene encoding the enzyme of interest.
• Gene expression can be decreased by, for example, modifying an expression control sequence of the gene such as promoter and the like.
• an expression control sequence preferably not less than one nucleotide, more preferably not less than 2 nucleotides, particularly preferably not less than 3 nucleotides in the expression control sequence are modified.
• the expression control sequence may be partly or entirely deleted.
• a modification to lower the enzyme activity can be achieved by, for example, partly or entirely deleting the coding region of a gene encoding the enzyme of interest on the chromosome.
• the whole gene including the sequences before and after the gene on the chromosome may be deleted.
• the region to be deleted may be any region such as N-terminal region, internal region, C-terminal region and the like as long as the enzyme activity can be decreased. Generally, a longer region to be deleted can certainly inactivate the gene. It is preferable that the sequences before and after the region to be deleted do not have the same reading frame.
• a modification that lowers the enzyme activity can also be achieved by, for example, introducing other sequence into the coding region of a gene encoding the enzyme of interest on the chromosome. While the insertion site may be any region of the gene, a longer region to be inserted can certainly inactivate the gene. It is preferable that the sequences before and after the region to be inserted do not have the same reading frame. While other sequence is not particularly limited as long as it decreases the function of the protein to be encoded or makes the function disappear, for example, a marker gene and a gene useful for the production of ⁇ -glutamyl compounds such as glutathione and the like can be mentioned.
• Modification of a gene on the chromosome as mentioned above can be achieved by, for example, producing a deleted gene lacking a partial sequence of the gene and modified to not produce a protein that functions normally, transforming a yeast with a recombinant DNA containing the deleted gene, and substituting the gene on the chromosome by a deleted gene by causing homologous recombination between the deleted gene and the gene on the chromosome.
• the operation is facilitated when the recombinant DNA contains a marker gene according to the phenotype of the host such as auxotrophy and the like.
• a strain having a recombinant DNA integrated with the chromosome can be efficiently obtained when the aforementioned recombinant DNA is linearized by cleavage with a restriction enzyme and the like. Even when a protein encoded by the deleted gene is produced, it has a steric structure different from that of a wild-type protein and the function thereof decreases or disappears.
• homologous recombination sometimes results in the insertion of a wild-type gene and a deleted gene into the chromosome, with the other part of the recombinant DNA (e.g., vector part and marker gene) interposed between them. Since the wild-type gene functions in this state, it is necessary to cause homologous recombination again between the two genes, remove one copy of the gene together with the vector part and the marker gene from the chromosome DNA, and select a sequence maintaining the deleted gene.
• the other part of the recombinant DNA e.g., vector part and marker gene
• a yeast is transformed with a linear DNA containing any sequence provided with, on both ends thereof, upstream and downstream sequences of the substitution target site on the chromosome, to cause homologous recombination at each of the upstream and downstream of the substitution target site, whereby the substitution target site can be substituted by said any sequence in one step.
• a sequence containing a marker gene can be used.
• the marker gene may be removed thereafter where necessary.
• a sequence for homologous recombination may be added to the both ends of the marker gene to enable efficient removal of the marker gene.
• Decrease in the enzyme activity of interest can be confirmed by measuring the activity of the enzyme.
• the activity of glutathione reductase can be measured by a known method (Glutathione Reductase Assay Kit Product No. 7510-100-K manufactured by Cosmo Bio etc.).
• Decrease of the transcription amount of a gene encoding the enzyme of interest can be confirmed by comparing the amount of mRNA transcribed from the gene with that of the parent strain.
• a method for evaluating the amount of mRNA Northern hybridization, RT-PCR and the like can be mentioned ( Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001 )).
• the amount of mRNA preferably decreases to, for example, not more than 50%, not more than 20%, not more than 10%, not more than 5%, or 0%, as compared to the parent strain.
• the amount of the enzyme of interest can be confirmed by Western blot using an antibody ( Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001 )).
• the amount of the enzyme of interest preferably decreases to, for example, not more than 50%, not more than 20%, not more than 10%, not more than 5%, or 0%, as compared to the parent strain.
• yeast transformation method As a transformation method of yeast, a method generally used for yeast transformation such as protoplast method, KU method ( H. Ito et al., J. Bateriol., 153-163 (1983 )), KUR method ( Fermentation and Industry, vol.43, p.630-637 (1985 )), electroporation method ( Luis et al., FEMS Micro biology Letters 165 (1998) 335-340 ), method using carrier DNA ( Gietz R.D. and Schiestl R.H., Methods Mol. Cell. Biol. 5:255-269 (1995 )) and the like can be adopted.
• KU method H. Ito et al., J. Bateriol., 153-163 (1983 )
• KUR method Fermentation and Industry, vol.43, p.630-637 (1985 )
• electroporation method Luis et al., FEMS Micro biology Letters 165 (1998) 335-340 )
• Thiol oxidase is generally an enzyme that catalyzes the reaction of the following formula (1) in vivo.
• thiol oxidase is known to be mainly involved in the folding of protein.
• ERV1 which is one kind of thiol oxidase, activates Mia40 by oxidizing a thiol group contained in Mia40 that introduces a disulfide bond into proteins in the mitochondrial inner membrane
• ERV1 which is one kind of thiol oxidase
• ERV1 activates Mia40 by oxidizing a thiol group contained in Mia40 that introduces a disulfide bond into proteins in the mitochondrial inner membrane
• Thiol oxidase in the present invention only needs to have, as mentioned above, an activity to oxidize an intermolecular or intramolecular thiol group of protein or peptide with an oxygen molecule and form a disulfide bond (i.e., thiol oxidase activity), and is not particularly limited. For example, it is preferably any of the following (a) - (f):
• a protein having the amino acid sequence shown by SEQ ID NO: 1 or 2, wherein 1 or plural amino acids are substituted, inserted, deleted and/or added can be prepared according to a known method described in " Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1989 )" and the like, and is encompassed in the above-mentioned protein as long as it has a thiol oxidase activity.
• amino acid sequence modified by substitution, insertion, deletion and/or addition may contain only one kind (e.g., substitution) of modification, or two or more kinds of modifications (e.g., substitution and insertion).
• an amino acid used for substitution is preferably an amino acid having properties similar to those of amino acid before substitution (cognate amino acid).
• amino acids in the same group in the following groups are the cognate amino acids.
• the plural amino acids mean, for example, not more than 60, preferably 20, more preferably 15, further preferably 10, further preferably 5, 4, 3 or 2 amino acids.
• sequence identity to the amino acid sequence shown in SEQ ID NO: 1 - 2 is preferably not less than 60%, more preferably not less than 70%, further preferably not less than 80%, further preferably not less than 85%, further preferably not less than 90%, most preferably not less than 95%.
• sequence identity to the amino acid sequence is expressed by a value obtained by comparing the amino acid sequence shown in SEQ ID NO: 1 - 2 and the amino acid sequence desired to be evaluated, dividing the number of positions, at which amino acids matched between the both sequences, by the total number of the compared amino acids, and multiplying the value by 100.
• An additional amino acid sequence can be bonded to the amino acid sequence described in SEQ ID NO: 1 - 2 as long as it has a thiol oxidase activity.
• a fusion protein with other protein can also be provided.
• the DNA that hybridizes with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown by SEQ ID NO: 3 or 4 under stringent conditions means a DNA obtained by colony hybridization method, plaque hybridization method, or Southern hybridization method and the like under stringent conditions by using a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown by SEQ ID NO: 3 or 4 as a probe.
• DNA that hybridizes under stringent conditions is, for example, a DNA obtained by hybridization using a filter immobilizing a colony or plaque-derived DNA in the presence of 0.7 - 1.0 M NaCl at 65°C, and washing the filter with 2-fold concentration of SSC solution (1 x concentration of SSC solution is composed of 150 mM sodium chloride and 15 mM sodium citrate) at 65°C.
• hybridization conditions are described above, they are not particularly limited to those conditions.
• factors affecting the stringency of hybridization plural factors such as temperature, salt concentration and the like are considered, and those of ordinary skill in the art can realize the optimal stringency by appropriately determining those factors.
• a DNA hybridizable under the above-mentioned conditions is, for example, a DNA having sequence identity of not less than 7.0%, preferably not less than 74%, more preferably not less than 79%, further preferably not less than 85%, further more preferably not less than 90%, most preferably not less than 95%, to the DNA shown by SEQ ID NO: 3 or 4.
• the sequence identity (%) of DNA is expressed by a value obtained by optimally aligning two DNAs to be compared, dividing the number of positions, at which a nucleic acid base (e.g., A, T, C, G, U or I) matched between the both sequences, by the total number of the compared nucleotides, and multiplying the resulting value by 100.
• a nucleic acid base e.g., A, T, C, G, U or I
• sequence identity of DNA can be calculated using, for example, the following tools for sequence analysis: GCG Wisconsin Package (Program Manual for The Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive Medison, Wisconsin, USA 53711; Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 1RQ, England) and the.e.xPASy World Wide Web Molecule Server for Biology (Geneva University Hospital and University of Geneva, Geneva, Switzerland).
• DNA having a nucleotide sequence shown by SEQ ID NO: 3 or 4 wherein 1 or plural nucleotides are substituted, deleted, inserted and/or added can be prepared according to a known method described in " Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1989 )" and the like.
• a nucleotide sequence modified by substitution, insertion, deletion and/or addition may contain only one kind (e.g., substitution) of modification, or two or more kinds of modifications (e.g., substitution and insertion).
• the plural nucleotides described above are not particularly limited as long as a protein encoded by the DNA has a thiol oxidase activity, and mean, for example, not more than 150, preferably 100, more preferably 50, further preferably 20, further more preferably 10, 5, 4, 3 or 2 nucleotides.
• the representative thiol oxidase in the present invention includes thiol oxidase (ERV1) and thiol oxidase (ERO1).
• Glutathione reductase is an enzyme having an activity to reduce oxidized glutathione represented by the following formula (2) by utilizing NADPH (reduced nicotinamide dinucleotide phosphate).
• NADPH reduced nicotinamide dinucleotide phosphate
• a protein having the amino acid sequence shown by SEQ ID NO: 5, wherein 1 or plural amino acids are substituted, inserted, deleted and/or added can be prepared according to the method described in the above-mentioned (1), and is encompassed in the above-mentioned protein as long as it has a glutathione reductase activity.
• amino acid sequence modified by substitution, insertion, deletion and/or addition may contain only one kind (e.g., substitution) of modification, or two or more kinds of modifications (e.g., substitution and insertion).
• an amino acid used for substitution is preferably an amino acid having properties similar to those of amino acid before substitution (cognate amino acid). Cognate amino acid is as mentioned above in (1).
• the plural amino acids mean, for example, not more than 60, preferably 20, more preferably 15, further preferably 10, further preferably 5, 4, 3 or 2 amino acids.
• sequence identity to the amino acid sequence shown in SEQ ID NO: 5 is preferably not less than 60%, more preferably not less than 70%, further preferably not less than 80%, further preferably not less than 85%, further preferably not less than 90%, most preferably not less than 95%.
• sequence identity to the amino acid sequence can be calculated by the aforementioned method in (1).
• An additional amino acid sequence can be bonded to the amino acid sequence described in SEQ ID NO: 5 as long as it has a glutathione reductase activity.
• a fusion protein with other protein can also be provided.
• the DNA that hybridizes with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown by SEQ ID NO: 6 under stringent conditions means a DNA obtained by colony hybridization method, plaque hybridization method, or Southern hybridization method and the like under stringent conditions by using a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown by SEQ ID NO: 6 as a probe.
• the conditions and the like of hybridization are as mentioned above in (1).
• a DNA hybridizable under the above-mentioned conditions is, for example, a DNA having sequence identity of not less than 70%, preferably not less than 74%, more preferably not less than 79%, further preferably not less than 85%, further more preferably not less than 90%, most preferably not less than 95%, to the DNA shown by SEQ ID NO: 6.
• the sequence identity (%) of the DNA is as mentioned in (1) above.
• DNA having a nucleotide sequence shown by SEQ ID NO: 6 wherein 1 or plural nucleotides are substituted, deleted, inserted and/or added can be prepared according to the aforementioned method in (1).
• a nucleotide sequence modified by substitution, insertion, deletion and/or addition may contain only one kind (e.g., substitution) of modification, or two or more kinds of modifications (e.g., substitution and insertion).
• the plural nucleotides described above are not particularly limited as long as a protein encoded by the DNA has a glutathione reductase activity, and mean, for example, not more than 150, preferably 100, more preferably 50, further preferably 20, further more preferably 10, 5, 4, 3 or 2 nucleotides.
• a means for confirming that a protein having the amino acid sequence shown by SEQ ID NO: 5, wherein 1 or plural amino acids are deleted, substituted, inserted and/or added, is a protein having a glutathione reductase activity is, for example, a method including producing a transformant that expresses a protein, whose activity is desired to be confirmed, by a DNA recombinant method, producing the protein by using the transformant, placing the protein, oxidized glutathione and NADPH in an aqueous medium, and analyzing the presence or absence of production and accumulation of reduced glutathione or NADP in the aqueous medium by HPLC and the like.
• the yeast of the present invention may heterozygously have a gene modified to show a decreased enzyme activity and a wild-type gene as long as it can accumulate ⁇ -glutamyl compounds such as glutathione and the like. However, it is generally preferably a homozygote of a gene modified to show a decreased enzymatic activity.
• the glutathione reductase activity is preferably decreased to not more than 50%, more preferably not more than 20%, further preferably not more than 10%, particularly preferably not more than 5%, as compared to the parent strain. Substantial disappearance of the glutathione reductase activity is preferable.
• ⁇ -glutamylcysteine synthetase in the present invention only needs to have an activity to condense glutamic acid and cysteine to synthesize glutamylcysteine (i.e., ⁇ -glutamylcysteine synthetase activity) and is not particularly limited,
• a protein having the amino acid sequence shown by SEQ ID NO: 7, wherein 1 or plural amino acids are substituted, inserted, deleted and/or added can be prepared according to the method described in the above-mentioned (1), and is encompassed in the above-mentioned protein as long as it has a ⁇ -glutamylcysteine synthetase activity.
• amino acid sequence modified by substitution, insertion, deletion and/or addition may contain only one kind (e.g., substitution) of modification, or two or more kinds of modifications (e.g., substitution and insertion).
• an amino acid used for substitution is preferably an amino acid having properties similar to those of amino acid before substitution (cognate amino acid). Cognate amino acid is as mentioned above in (1).
• the plural amino acids mean, for example, not more than 60, preferably 20, more preferably 15, further preferably 10, further preferably 5, 4, 3 or 2 amino acids.
• sequence identity to the amino acid sequence shown in SEQ ID NO: 7 is preferably not less than 60%, more preferably not less than 70%, further preferably not less than 80%, further preferably not less than 85%, further preferably not less than 90%, most preferably not less than 95%.
• sequence identity to the amino acid sequence can be calculated by the aforementioned method in (1).
• An additional amino acid sequence can be bonded to the amino acid sequence described in SEQ ID NO: 7 as long as it has a ⁇ -glutamylcysteine synthase activity.
• a fusion protein with other protein can also be provided.
• the DNA that hybridizes with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown by SEQ ID NO: 8 under stringent conditions means a DNA obtained by colony hybridization method, plaque hybridization method, or Southern hybridization method and the like under stringent conditions by using a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown by SEQ ID NO: 8 as a probe.
• the conditions and the like of hybridization are as mentioned above in (1).
• a DNA hybridizable under the above-mentioned conditions is, for example, a DNA having sequence identity of not less than 70%, preferably not less than 74%, more preferably not less than 79%, further preferably not less than 85%, further more preferably not less than 90%, most preferably not less than 95%, to the DNA shown by SEQ ID NO: 8.
• the sequence identity (%) of the DNA is as mentioned in (1) above.
• DNA having a nucleotide sequence shown by SEQ ID NO: 8 wherein 1 or plural nucleotides are substituted, deleted, inserted and/or added can be prepared according to the aforementioned method in (1).
• a nucleotide sequence modified by substitution, insertion, deletion and/or addition may contain only one kind (e.g., substitution) of modification, or two or more kinds of modifications (e.g., substitution and insertion).
• the plural nucleotides described above are not particularly limited as long as a protein encoded by the DNA has a ⁇ -glutamylcysteine synthetase activity, and mean, for example, not more than 150, preferably 100, more preferably 50, further preferably 20, further more preferably 10, 5, 4, 3 or 2 nucleotides.
• various inorganic and organic ammonium salts such as ammonia or ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium acetate and the like, urea and other nitrogen-containing compounds, as well as nitrogenous organic substances such as peptone, NZ amine, meat extract, yeast extract, corn steep liquor, casein hydrolysate, fish meal or digest thereof, defatted soybean or digest and hydrolysate thereof, and the like, and various amino acids such as aspartic acid, glutamic acid, threonine and the like can be used.
• ammonia or ammonium chloride ammonium phosphate, ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium acetate and the like
• nitrogenous organic substances such as peptone, NZ amine, meat extract, yeast extract, corn steep liquor, casein hydrolysate, fish meal or digest thereof, defatted soybean or
• the yeast culture can be optimized and the final cell concentration and the activity of glutathione synthesis-related enzymes can be improved, as a result of which the producibility of glutathione can be actually improved.
• glutathione containing a reduced type at a higher proportion by once reducing oxidized glutathione contained in the above-mentioned glutathione extract or the glutathione powder.
• reduction method is not particularly limited, reduction by glutathione reductase is preferably mentioned.
• genomic DNA of Saccharomyces cerevisiae YPH499 strain as a template and 5'-GGCCGCTAGCATGAAAGCAATAGATAAAATGACGG-3' (SEQ ID NO: 13) and 5'-GGCCGGATCCTTATTCGTCCCAGCCGTCCTTCC-3' (SEQ ID NO: 14) as primers, PCR amplification was performed, and the amplification product was digested with NheI and BamHI to give a thiol oxidase (ERV1) gene NheI-BamHI fragment. The fragment was ligated to the NheI-BamHI digestion site of pGK406 described in a non-patent document ( J. Biochem.
• ERV1-expression plasmid pGK406-ERV1 was digested with restriction enzyme NcoI.
• Saccharomyces cerevisiae YPH499/GSH1, GSH2 strain shown in Production Example 1 as host yeast strain was transformed to give transformant YPH499/GSH1, GSH2, ERV1 strain (GSH1-enhanced + GSH2-enhanced + ERV1-enhanced strain).
• genomic DNA of Saccharomyces cerevisiae YPH499 strain as a template and 5'-GGCCGCTAGCATGAGATTAAGAACCGCCATTGCCAC-3' (SEQ ID NO: 15) and 5'-GGCCGGATCCTTATTGTATATCTAGCTTATAGGAAATAGGC-3' (SEQ ID NO: 16) as primers, PCR amplification was performed, and the amplification product was digested with NheI and BamHI to give a thiol oxidase (ERO1) gene NheI-BamHI fragment. The fragment was ligated to the NheI-BamHI digestion site of pGK406 described in a non-patent document ( J. Biochem.
• Saccharomyces cerevisiae YPH499/ ⁇ GLR1, GSH1, GSH2 strain, in which expression of GSH1 gene and GSH2 gene is enhanced, and GLR1 gene was destructed was obtained by the method described in a non-patent document ( Kiriyama K, Hara KY, Kondo A. (2013) Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism. ApplMicrobiol Biotechnol, 97(16):7399-7404 .).
• genomic DNA of Saccharomyces cerevisiae YPH499 strain as a template and 5'-AAAAGGATCCATGGCTGGTAATCTTGTTTCATGGGCC-3' (SEQ ID NO: 17) and 5'-AAAACTCGAGTTAATTTTCATTGACCAAACCAGCCTCC-3' (SEQ ID NO: 18) as primers, PCR amplification was performed, and the amplification product was digested with BamHI and XhoI to give a glutathione transport enzyme (YCF1) gene BamHI-XhoI fragment.
• YCF1 glutathione transport enzyme
• Example 1 Production of glutathione by yeast in which expression of GSH1 gene and GSH2 gene is enhanced, and expression of ERV1 gene or ERO1 gene is further enhanced (GSH1-enhanced + GSH2-enhanced + ERV1-enhanced strain, GSH1-enhanced + GSH2-enhanced +ERO1-enhanced strain)
• SD medium 6. g/L yeast nitrogen base w/o amino acids (manufactured by Difco laboratories), 20 g/L glucose) (5 ml) at 30°C for 16 - 24 hr.
• YPD medium 10 g/L yeast extract
• the glutathione content was calculated by dividing the above-mentioned glutathione concentration by the cell concentration to give glutathione weight per dry cell (glutathione content).
• Example 2 Production of glutathione by strain in which expression of GSH1 gene and GSH2 gene is enhanced, expression of GLR1 gene is decreased, and expression of ERV1 gene or ERO1 gene is further enhanced (GSH1-enhanced + GSH2-enhanced + GLR1-destructed + ERV1-enhanced strain, GSH1-enhanced + GSH2-enhanced + GLR1-destructed + ERO1-enhanced strain)
• SD medium 6.
• yeast extract manufactured by Difco laboratories
• polypeptone manufactured by Wako Pure Chemical Industries, Ltd.
• glucose manufactured by Nacalai Tesque
• Example 3 Production of glutathione by strain in which ERV1 is enhanced, expression of GSH1, GSH2 and YCF1 is further enhanced, and GLR1 gene is destructed (GGSH1-enhanced + GSH2-enhanced + ERV1-enhanced + YCF1-enhanced + GLR1-destructed strain)

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